Introduction: Heme solution employed was obtained from sterile bovine blood, using physical and chemical methods. To characterize it, is necessary to separate molecules by size within it. Therefore the aim of this study was to design a method for separating the different fractions present in said solution Hemo (PM-616 Da) and characterize them with spectroscopic techniques. Material and Methods: Dialysis membranes 1 000 Da, 6-8000 Da and 15,000 Da and centrifuge tubes Amicon membrane 10 000 Da were used. Two lots of hemo solution adjusted to pH 4.4 and 5.0 were used. The dialyzed solution was treated with nitric acid (HNO3) and 86% concentrated to 20 mL to determine iron by atomic absorption (AA). Dialysis was performed with 2 L of distilled water and concentrated to 10 mL heat, also the case with HNO3. Purity was determined by mass spectrometry (MS) being used as a standard swine hematin (PM-633.9 Da, Sigma). Results: The hydrolyzated to pH 4.4 achieved the best results with a 82.32% with molecules smaller size 1000 Da, 2.72% between 1000 and 6-8 000 Da, 4.24% between 6 – 8 000 and 15 000 Da and 15 000 Da above 10.72%, which is attributed to form aggregates where the heme is present in monomeric form was obtained. The purity and protein concentration is presented in the results obtained by MS.